Overexpression of estrogen receptor alpha (ERalpha) transcriptional regulators, such as AIB1 (coactivator amplified in breast cancer-1), has been demonstrated in many ERalpha-positive human breast cancer cell lines and tumors. Many transcriptional coactivators, including members of the Steroid Receptor Coactivator-1 family (SRC-1, GRIP1/TIF1/SRC-2, c/CIP/AIB1/SRC-3), can potentiate the estrogen-induced transcriptional activation of ERalpha target genes in transfection experiments. These findings, together with our recent data that mammary epithelial cells expressing SRC-1 are independent of these expressing ERalpha in normal breast, suggest a role for ERalpha coregulators in estrogen-stimulated breast cancer cell proliferation. The contributions of ERalpha coregulators in breast biology and estrogen-mediated cell proliferation are not clear. We postulate that overexpression of SRC-1 family members contributes to estrogen-stimulated breast cancer cell proliferation. Our working hypotheses are: 1) SRC-1 family members act as coactivators for ERalpha and are segregated from ERalpha in normal epithelium; and 2) overexpression and colocalization of SRC-1 family members with ERalpha potentiates the transactivation function of ERalpha and estrogen-stimulated cancer cell proliferation. To integrate knowledge of cell signaling with whole organ biology, three aims are proposed to test the above hypotheses. In Aim 1, we will profile the expression and localization of ERalpha and SRC-1 family members during mammary gland development and compare this to carcinogen-induced mammary tumors. The coactivators which are overexpressed in tumors, or segregated from ERalpha in normal mammary epithelium but colocalized with ERalpha in breast tumors, will be used for subsequent studies. In Aim 2, we will assess if overexpression or altered colocalization of SRC-1 family members can enhance in situ ERalpha transactivation function in normal breast. We will use our unique adenoviral approach to reconstitute ERalpha transactivation function in situ and deliver genes of interest locally into mammary epithelium in an intact microenvironment. In Aim 3, we will assess the contribution of SRC-1 family members to ERalpha transactivation function in modulating estrogen-stimulated cell proliferation and development of the mammary gland. These studies will provide a better understanding of the roles of transcriptional regulators of ERalpha in estrogen-regulated cell proliferation. The identification of abnormal expression and localization of these transcriptional regulators may lead to the development of new therapeutic targets and improved strategies for the prevention and treatment of breast cancer.